ABSTRACT
Cryopreservation of mesenchymal stem cells from amniotic fluid is of clinical importance, as these cells can be harvested during the prenatal period and stored for use in treatments. We examined the behavior of mesenchymal stem cells from human amniotic fluid in culture that had been subjected to cryopreservation. We assessed chromosomal stability through karyotype analysis, determined whether multipotent capacity (differentiation into adipogenic, chondrogenic, and osteogenic cells) is maintained, and analyzed SOX2 and NANOG expression after thawing. Five amniotic fluid samples were cryopreserved for 150 days. No chromosomal aberrations were observed. The expression levels of NANOG and SOX2 also were quite similar before and after cryopreservation. Capacity for differentiation into adipogenic, chondrogenic, and osteogenic tissues also remained the same. We conclude that cryopreservation of amniotic fluid does not alter karyotype, NANOG/SOX2 gene expression, or multipotent capacity of stem cells that have been collected from amniotic fluid during pregnancy.
Subject(s)
Amniotic Fluid/metabolism , Cryopreservation , Homeodomain Proteins/genetics , Karyotyping , Mesenchymal Stem Cells/metabolism , SOXB1 Transcription Factors/genetics , Amniotic Fluid/cytology , Base Sequence , Cell Differentiation , DNA Primers , Female , Flow Cytometry , Gene Expression , Humans , Mesenchymal Stem Cells/cytology , Nanog Homeobox Protein , PregnancyABSTRACT
In view of the serious consequences of medium-chain acyl-CoA dehydrogenase (MCAD) deficiency and the absence of information about its incidence in the Brazilian population, we examined the frequency of the A985G mutation in the MCAD gene. A retrospective analysis was made of data on 1722 individuals (844 females) genotyped for the A985G mutation in the MCAD gene, using genomic DNA extracted from peripheral blood leukocytes and genotyping with PCR-RFLP; 0.41% of these individuals were heterozygous for the A985G mutation. The mutant homozygous genotype was not found. The 985G mutant and 985A normal alleles had allelic frequencies of 0.0020 and 0.9980, respectively. Given the A985G allele frequency, genotyping would be recommended in cases of family history of MCAD deficiency and sudden infant death syndrome, and when there is suspicion of medium-chain fatty acid metabolic alterations; genetic counseling should be offered in cases involving 985GG and A985G individuals and consanguineous marriages.
Subject(s)
Acyl-CoA Dehydrogenase/genetics , Gene Frequency/genetics , Mutation/genetics , Adolescent , Adult , Aged , Brazil , Child , Child, Preschool , Female , Genotype , Humans , Infant , Infant, Newborn , Male , Middle Aged , Polymerase Chain Reaction , Polymorphism, Restriction Fragment Length , Young AdultABSTRACT
Charcot-Marie-Tooth type 1A disease (CMT1A) is most frequently caused by a tandem DNA duplication of a 1.4-Mb genomic fragment in the 17p11.2-12 chromosomal region. The disease is probably the product of a dosage effect of the peripheral myelin protein 22 gene located within the duplicated segment. We sought to study the largest reported Brazilian family with suspected diagnosis of CMT1A using eight short tandem repeat microsatellite markers. In addition, we analyzed the informativeness of these markers in the normal Brazilian population. The duplication was found in 12 members of the family. In two patients with CMT1A symptoms, the duplication was not detected, and one asymptomatic subject showed the duplication. D17S2230, D17S9B, D17S2220, D17S2227, D17S9A, and D17S4A markers showed the highest heterozygosity rates, and D17S2228 and D17S2224 markers were the least informative in our analysis.
Subject(s)
Charcot-Marie-Tooth Disease/diagnosis , Microsatellite Repeats/genetics , Brazil , Charcot-Marie-Tooth Disease/genetics , Chromosomes, Human, Pair 17/genetics , Gene Duplication , Gene Frequency , Genetic Markers/genetics , Genetics, Population , Humans , Models, Genetic , Myelin Proteins/geneticsABSTRACT
A determinação do sexo do feto é geralmente realizada pelo procedimento de ultra-som que ocorre no segundo trimestre da gestação, sendo que, quando realizado antes da 13ª semana, esse método se mostra muito incerto.Técnicas moleculares utilizando o PCR já foram descrita, e possuem maior sensibilidade na determinação do sexo. Dentre estas técnicas existentes, as invasivas consistem em amniocentese ou coleta de amostra de vilo coriônico, seguida de PCR para determinar osexo e que representam em aumento de risco na gravidez e as técnicas não invasivas que conseguem detectar o DNA fetal no sangue periférico materno. Foi desenvolvida em nosso laboratório uma técnica capaz de detectar o sexo fetal nas primeiras semanas de gestaçãocom alto índice de acerto. A técnica consistiu em desenhar iniciadores que anelassem em regiões repetitivas espalhadas no cromossomoY e PCR Nested para aumentar a acurácia do exame. Os resultados demonstraram que a técnica possui sensibilidade e especificidade de 100%. Além disso, a PCR foi capaz de detectar em uma diluição seriada de uma amostra de DNA genômico masculina, previamente quantificada, a presença do cromossomo Y em amostrascontendo apenas 100 femtogramas de DNA.
The determination of fetal sex is currently carried out by ultrasound that is usually performed in the second trimester. However, it is inaccurate before 13th week of gestation. Molecular techniques such as PCR already had been described and were successful of fetal gender. Among these techniques, there are invasive methods: chorionic villus sampling and amniocentesis that are avoided because it is associated with a risk of fetal loss, and the non-invasive procedures that use of fetal DNA circulating in maternal blood. We report a new Nested PCR method with specific primers for repetitive sequences of the Y-chromossome. Our results indicated that sensitivity and specificity of the method were 100% and we can accurately detect Y-chromossome sequences in samples with only 100 femtograms of DNA template.
